NOTE: In the following, a protocol for geneti-chemical PEGylation of an Ad vector is described to detail. To enable specific coupling of the PEG moiety, an Ad5 vector was beforehand genetically modified by introducing a cysteine residue into the hexon protein at the hypervariable loop 5 as described in a previous publication36, and a maleimide-activated PEG compound is used as coupling compound.
1. Preparation of Buffers for Vector Purification by CsCL Step Gradients
<ol>
	<li>Prepare 400 mL of Adenovirus buffer (Ad-buffer) by adding 50 mM HEPES (4.76 g/400 mL) and 150 mM NaCl (3.504 g/400 mL) to double-distilled water (ddH2O). 350 mL of this buffer is needed to prepare the following three Ad-buffer variants.
	<ol>
		<li>Variant A: adjust 150 mL of Ad-buffer to pH 7.6 with NaOH.</li>
		<li>Variant B: add a final concentration of 10 mM TCEP [tris(2-carboxyethyl)phosphine), 0.143 g] to 50 mL and adjust it to pH 7.2 with HCl.</li>
		<li>Variant C: add a final concentration of 0.5 mM TCEP (0.022 g) to 150 mL and adjust it to pH 7.2 with HCl.</li>
		<li>Prepare the buffers in ddH2O and analytical grade chemicals. Use 100 mL each of variant A and variant C to prepare the CsCl buffers (see steps 1.2 and 1.3; 50 mL of each) necessary for the CsCl step gradients (see steps 3.2.6. and 3.2.18).</li>
	</ol>
	</li>
	<li>Preparing the CsCl step gradient buffer (&#961;CsCl = 1.41 g/cm3) in Ad-buffer 
	NOTE: This buffer will be needed in two different variations:
	<ol>
		<li>Weigh two aliquots of exactly 27.42 g of CsCl into 50 mL tubes.</li>
		<li>&#961;CsCl = 1.41/TCEP buffer: resolve CsCl in 40 mL of variant C of Ad-buffer.</li>
		<li>&#961;CsCl = 1.41 buffer: resolve CsCl in 40 mL of variant A of Ad-buffer.</li>
		<li>Check, and if necessary, adjust the pH with HCl. Fill up to exactly 50 mL with the corresponding Ad-buffer variant. To achieve the best separation results, the exact CsCl concentration and pH are crucial.</li>
		<li>Check the density (CsCl content) by weighing 1 mL (1.41 g) of each gradient buffer.</li>
	</ol>
	</li>
	<li>Preparing the CsCl step gradient buffer (&#961;CsCl = 1.27 g/cm3) in Ad-buffer 
	NOTE: This buffer is needed in two different variations:
	<ol>
		<li>Weigh two aliquots of exactly 18.46 g of CsCl in 50 mL tubes.</li>
		<li>&#961;CsCl = 1.27/TCEP buffer: resolve CsCl in 40 mL of variant C of Ad-buffer.</li>
		<li>&#961;CsCl = 1.27 buffer: resolve CsCl in 40 mL of variant A of Ad-buffer.</li>
		<li>Check, and if necessary, adjust the pH with HCl. Finally, fill up to exactly 50 mL with the corresponding Ad-buffer variant. To achieve the best separation results, the exact CsCl concentration and pH are crucial.</li>
		<li>Check the density (CsCl content) by weighing 1 mL (1.27 g) of each gradient buffer.</li>
	</ol>
	</li>
	<li>Sterilize all buffers (Ad-buffers and CsCl step gradient buffers) by filtration (using 0.45 &#181;m mesh pore size) into sterile bottles.</li>
	<li>After sterilization, to prevent oxidation of TCEP, saturate and overlay the buffers containing TCEP with argon by sparging the buffers with argon gas for about 30 s. Take care to only use sterile devices (e.g., sterile pipette tips) when applying the argon gas, and use bottles large enough to allow argon blubber. 
	NOTE: All buffers should be prepared fresh and protected from light and can be stored at 4 &#176;C for several days (especially the CsCl step gradient buffers that should only be kept for a few days).</li>
</ol>
2. Coupling Moieties: Storage and Preparation
NOTE: Moeities used for coupling to cysteines need to bear thiol-reactive groups. Maleimide-activated compounds will form stable thioether bonds with the genetically introduced cysteines. Alternatively, ortho-pyridyldisulfide (OPSS)-activated compounds can be used, which form bioresponsive disulfide bridges between the vector particles and coupling moiety. Lyophilized malPEG-750 as well as most other coupling reagents are sensitive to hydrolysis and should be stored dry in the form of lyophilized powders at -80 &#176;C.
<ol>
	<li>To prevent the build-up of condensing water upon thawing of the vials, store the vials in larger tubes (e.g., 50 mL tubes) filled with a cushion of silica gel beads. Store these tubes in an adequate larger container also filled with a silica gel bead cushion.</li>
	<li>Before tightly closing each tube and container, exchange air with argon gas. This can be achieved by placing the open tubes and containers into a desiccator, followed by removing and replacing the air with argon gas. Since argon gas is heavier than air, tubes and containers are filled up with argon gas and can now be placed into each other, with each lid tightly closed.</li>
	<li>Store containers at -80 &#176;C.</li>
	<li>When thawing, place the containers onto silica beads in a desiccator and slowly warm them up to room temperature, then open the container.</li>
	<li>When performing a coupling reaction, freshly resolve the amount of coupling substrate needed into the appropriate solvent. Take care not to add more than 10% of coupling solution to the final coupling reaction, especially if DMF or DMSO is used as the solvent for the coupling substrate.</li>
</ol>
3. Amplification, Purification and Chemical Modification of Ad Vectors:
<ol>
	<li>Amplification of Ad vectors 
	NOTE: The following steps must be performed using a safety cabinet according to local biological safety rules.
	<ol>
		<li>Transfect a replication deficient &#8710;E1 Ad vector containing one (or several) genetically introduced cysteine residue(s) into HEK 293 cells as described by Kratzer and Kreppel38.</li>
		<li>According to the protocol38, amplify the vector by sequential reinfections up to a final preparative infection of 15-20 large (15 cm) cell culture plates (approximately 1-2 x 107 cells/plate). 
		NOTE: Optimally, the last reinfection should be timed so that cells show full cytopathic effect (CPE) on the morning of purification and modification performance (see Figure 2).</li>
		<li>After the final amplification step, cells resuspended in Ad buffer + 10 mM TCEP (variant B) can be frozen at -80 &#176;C; however, for optimal yield of vector particles, an immediate follow-up of cell lysis and vector purification and modification is recommended.</li>
	</ol>
	</li>
	<li>Purification and chemical modification of Ad vectors 
	NOTE: Purification of vectors is performed according to the adenovirus purification protocol described in38 but under non-oxidizing conditions. Cell harvesting and lysis as well as vector purification and chemical modification can be performed within one day. Harvest and lyse the infected cells according to the protocol described previously38.
	<ol>
		<li>Collect cells by scraping the plates and transferring the supernatant to 200-500 mL centrifugation tubes.</li>
		<li>Spin the cell solution for 10 min at 400 x g.</li>
		<li>Resuspend the cell pellet in 4 mL of Ad-buffer + 10 mM TCEP (pH 7.2) (variant B) and transfer to a sterile 50 mL tube. If cell yield is low or only a weak CPE was induced, resuspend it in 2 mL.</li>
		<li>Rescue the vector by 3 cycles of freezing (in liquid nitrogen) and thawing (in a 37 &#176;C water bath).</li>
		<li>Spin for 10 min at 5,000 x g at 4 &#176;C.</li>
		<li>While centrifuging, prepare two equal CsCl step gradients:
		<ol>
			<li>First, as the lower phase, add 3 mL of 1.41 g/cm3 &#961;CsCl in Ad-buffer + 0.5 mM TCEP (pH 7.2, variant C) into the ultracentrifuge tubes. Mark the level of the lower phase on the tube before loading the upper phase.</li>
			<li>Carefully stack the upper phase of 5 mL of 1.27 g/cm3 &#961;CsCl in Ad-buffer + 0.5 mM TCEP (pH 7.2, variant C) by very slowly pipetting the 5 mL volume along the walls of the tube onto the lower phase. 
			NOTE: Since during coupling a high concentration of TCEP could react with the maleimide residue of malPEG-750 and lead to reduced coupling efficiency, purification by CsCl step gradient needs to be already performed under a reduced TCEP concentration.</li>
		</ol>
		</li>
		<li>Load the supernatant onto the CsCl gradient [either equally divide the supernatant onto both gradients or, in the case of a low vector yield (see above), load the supernatant onto only one column], and fill both gradients equally to the top with Ad buffer + 10 mM TCEP (pH 7.2, variant B).</li>
		<li>Adjust the weight of the opposite tubes to a 0.0 g weight difference.</li>
		<li>Ultracentrifuge for 2 h at 176,000 x g at 4 &#176;C.</li>
		<li>With a clamp, fix the ultracentrifugation tube to a stand, leaving the area around the lower phase level mark accessible. Place the gooseneck lamp above the tube. Around the mark, at the border between the lower and upper phases, a distinct band (the vector virions) should be visible (Figure&#160;3A-3C).</li>
		<li>Collect the vectors by puncturing the tube with a needle and aspiring the vector band into a syringe. Transfer collected vector virions to a 15 mL tube. 
		NOTE: Weaker bands above the Ad vector band contain incomplete vector particles and should be avoided for aspiration. The cell debris and green fluorescent protein (EGFP) of EGFP-expressing Ad-vectors will collect in the upper part of the ultracentrifuge tube (see Figure&#160;3A and 3B). This and the following steps up to coupling (step 3.2.16) should be performed speedily, as the vectors are now sensible to disulfide bonding due to the low TCEP concentration.</li>
		<li>To calculate the amount of coupling substrate needed for efficient complete binding of the substrate to all cysteine residues in the vector preparation, measure OD260:
		<ol>
			<li>According to the protocol described by Kratzer and Kreppel38, vortex a mixture of 10 &#181;L of the collected vector virions, 89 &#181;L of Ad buffer, and 1 &#181;L of 10% SDS. As a blank probe, vortex 99 &#181;L of Ad-buffer and 1 &#181;L of 10% SDS.</li>
			<li>To denature the virions, incubate both at 56 &#176;C for 10 min.</li>
			<li>Determine OD260.</li>
			<li>Calculate the physical vector titer per &#181;L using the following equation: OD260 x Factor of dilution x Empirically determined extinction coefficient of Ad (1.1 x 109 vector particles = 1 OD260 unit/&#181;L). Therefore, a measured OD260 of 1.36 of a vector diluted 1:10, would calculate to: 1.36 x 10 x 1.1 x 109 = approximately 1.5 x 1010 vector particles/&#181;L. 
			NOTE: This titer is only a rough estimation; however, it is accurate enough for the stoichiometric calculations outlined in the following.</li>
		</ol>
		</li>
		<li>Depending on the protein modified by introduction of a cysteine, determine the amount of coupling substrate (in grams) for an efficient coupling reaction, according to the following general equation: Virus titer x Volume x Number of cysteines x Excess of moiety x Molecular weight of moiety / 6,022 x 1023 = grams of coupling substrate. 
		NOTE: In this equation: 1) virus titer is the titer/&#181;L, determined by OD260 as described above, 2) volume accounts for the volume in &#181;L of aspired vector used in the coupling reaction, 3) the number of cysteines is the number of proteins into which the cysteine residue was introduced per vector particle, 4) excess of moiety is the factor of moiety excess necessary for an efficient coupling reaction (50x), and 5) the molecular weight of moiety must be introduced as Da (not kDa).</li>
		<li>Freshly resolve the amount of compound (or feasible amount) needed in the appropriate solvent. 
		NOTE: As the amount of coupling substrate needed is low and difficult to weigh but expensive, weigh the minimal amount of compound possible and dissolve it in an appropriate concentration for application to the coupling reaction.</li>
		<li>Transfer the vector solution to an appropriate size tube (e.g., 2 mL tube) and add the calculated amount of coupling compound stock solution to the vector.</li>
		<li>Gently mix the solution by overhead rotation for 1 h at room temperature.</li>
		<li>Fill up the vector solution to 6 mL total volume with Ad-buffer (pH 7.6, variant A). 
		NOTE: This step must be performed before the solution is applied to the step gradient to ensure that the vector solution, which still contains CsCl from the first step gradient, has a density below 1.27 g/mL.</li>
		<li>In a second CsCl banding step, purify the vector from the unreacted coupling moiety:
		<ol>
			<li>Prepare two equal CsCl step gradients by performing the following steps for each: 1) Lower phase: 3 mL of 1.41 g/cm3 &#961;CsCl in Ad-buffer (pH 7.6, variant A), 2) mark level of lower phase on the tube before loading upper phase, and 3) upper phase: 5 mL of 1.27 g/cm3 &#961;CsCl in Ad-buffer (pH 7.6, variant A). 
			NOTE: After coupling has taken place, buffers without TCEP are used, as no free thiol groups should now be present on the vector capsids.</li>
			<li>Load the supernatant onto the CsCl gradient and fill both gradients equally to the top with Ad-buffer (pH 7.6, variant A).</li>
			<li>Adjust the weights of the opposite tubes to a 0.0 g weight difference.</li>
		</ol>
		</li>
		<li>Ultracentrifuge for 2 h at 176,000 x g at 4 &#176;C.</li>
		<li>Shortly before the end of ultracentrifugation, equilibrate a PD-10 column 5 times with 5 mL of Ad-buffer (pH 7.6, variant A).</li>
		<li>As done in step 3.2.10, fix the ultracentrifugation tube to stand leaving area around the lower phase level mark accessible. Place the gooseneck lamp above the tube. Again, around the border between the lower and upper phases, a distinct band (the vector virions) should be visible (Figure 3C).</li>
		<li>Collect the vectors by puncturing the tube with a needle and aspiring the vector band into a syringe, and transfer the vector to a 15 mL tube. Take care to collect the virions of both gradient tubes together as a 2.5 mL total volume or less. If a smaller volume is collected, fill to obtain a 2.5 mL total volume with Ad buffer (variant A).</li>
		<li>Load the 2.5 mL onto the equilibrated PD-column. Discard the flow-through.</li>
		<li>Add 3 mL of Ad buffer (variant A) onto the column and collect the flow-through (containing the purified vector virions).</li>
		<li>Add glycerol (333 &#181;L) to obtain a final concentration of 10% and divide into suitable aliquots (e.g., 400 &#181;L each), and include an aliquot of 20 &#181;L for titer determination by OD260 measurement.</li>
		<li>Store the vector solution at -80 &#176;C.</li>
		<li>Determine the titer of vector by measuring the OD260 of 20 &#181;L vector solution, 79 &#181;L of Ad-buffer (variant A), and 1 &#181;L of 10% SDS as described in step 3.2.12. As a blank, use 79 &#181;L of Ad-buffer (variant A), 10 &#181;L of 10% glycerol, and 1 &#181;L of 10% SDS.</li>
		<li>Calculate the vector titer as: OD260 x Factor of dilution x 1.1 x 109 vector particles/&#181;L. 
		As prepared in step 3.2.27, calculate: OD260 x 5 x 1.1 x 109 vector particles/&#181;L</li>
	</ol>
	</li>
</ol>
4. Verification of Coupling by SDS-PAGE:
NOTE: If compounds with sufficiently high molecular weights are used for coupling to Ad virions, coupling can be verified by polyacrylamide gel electrophoresis (SDS-PAGE). Successful coupling should then result in a shift of the protein band corresponding to the modified Ad virion protein, compared to the protein in the unmodified Ad virion (see Figure 4).
<ol>
	<li>According to the calculated titer of vector (step 3.2.28), separate 1-2 x 1010 vector particles by SDS-PAGE (8%).</li>
	<li>Develop the gel by silver-staining of the gel39 to detect even weak protein bands:
	<ol>
		<li>Prepare buffers for silver staining (the amounts needed for 100 mL of buffer are indicated in brackets):
		<ol>
			<li>Prepare buffer 1 by mixing 38 mL of ddH2O, 50% MeOH (50 mL of 100 MeOH), 12% AcOH (12 mL of 100% AcOH), and 0.0185% HCHO (50 &#181;L of 37% HCHO).</li>
			<li>Prepare buffer 2 by adding 50% EtOH (50 mL of 100% EtOH) to 50 mL of ddH2O.</li>
			<li>Prepare buffer 3 by adding 0.127 g/L Na2S2O3 (12.744 mg) to 100 mL of ddH2O.</li>
			<li>Prepare buffer 4 by adding 2 g/L AgNO3 (0.2 g) and 0.0185% HCHO (50 &#181;L of 37% HCHO) to 100 mL of ddH2O.</li>
			<li>Prepare buffer 5 by adding 60 g/L Na2CO3 (6 g), 0.0185% HCHO (50 &#181;L of 37% HCHO), and 2.5 mg/L Na2S2O3 (0.256 mg) to ddH2O to obtain a final volume of 100 mL.</li>
			<li>Prepare buffer 6 by mixing 38 mL of ddH2O, 50% MeOH (50 mL of 100% MeOH), and 12% AcOH (12 mL of 100% AcOH).</li>
			<li>Prepare buffer 7 by mixing 60 mL of ddH2O, 30% MeOH (30 mL of 100% MeOH), and 10% glycerine (10 mL of 100% glycerine).</li>
			<li>Prepare buffer 8 by mixing 10% glycerine (10 mL of 100% glycerine) with 90 mL of ddH2O.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Performing silver staining
	<ol>
		<li>Fix the gel in buffer 1 for 30 min.</li>
		<li>Wash the gel in buffer 2 for 15 min.</li>
		<li>Pretreat the gel in buffer 3 for 1 min.</li>
		<li>Wash the gel 3 times in ddH2O for 20 s.</li>
		<li>Impregnate the gel in buffer 4 for 20 min.</li>
		<li>Wash the gel 2 times in ddH2O for 20 s.</li>
		<li>Develop the gel in buffer 5 until bands are visible (10 s to 10 min).</li>
		<li>Wash the gel 2 times in ddH2O for 2 min.</li>
		<li>Stop development in buffer 6 for 5 min.</li>
		<li>Conserve the gel in buffer 7 for 20 min.</li>
		<li>Store the gel in buffer 8. 
		NOTE: Coupling efficiency can be determined by densitometric quantification of the modified and unmodified bands of the targeted capsomere.</li>
	</ol>
	</li>
</ol>

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+serotype+5+hexon+mediates+liver+gene+transfer.">Adenovirus serotype 5 hexon mediates liver gene transfer.</a> Cell. 132, 397-409 (2008).</li><li>Kalyuzhniy, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+serotype+5+hexon+is+critical+for+virus+infection+of+hepatocytes+in+vivo.">Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo.</a> Proceedings of the National Academy of Sciences USA. 105, 5483-5488 (2008).</li><li>Vigant, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+hexon+hypervariable+region+5+of+adenovirus+serotype+5+abrogates+blood+factor+binding+and+limits+gene+transfer+to+liver.+Molecular+Therapy.">Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 16, 1474-1480 (2008).</li><li>Jonsson, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+factors+IX+and+X+enhance+binding+and+infection+of+adenovirus+types+5+and+31+in+human+epithelial+cells.">Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells.</a> Journal of Virology. 83, 3816-3825 (2009).</li><li>Bradshaw, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+receptor+engagement+during+infection+by+adenovirus+complexed+with+blood+coagulation+factor+X.">Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X.</a> PLoS Pathogen. 6, e1001142 (2010).</li><li>Duffy, M. R., Bradshaw, A. C., Parker, A. L., McVey, J. H., Baker, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cluster+of+basic+amino+acids+in+the+factor+X+serine+protease+mediates+surface+attachment+of+adenovirus/FX+complexes.">A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes.</a> Journal of Virology. 85, 10914-10919 (2011).</li><li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+factor+X+shields+adenovirus+type+5+from+attack+by+natural+antibodies+and+complement.">Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.</a> Nature Medicine. 19, 452-457 (2013).</li><li>Prill, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+of+adenovirus+hexon+allow+for+either+hepatocyte+detargeting+or+targeting+with+potential+evasion+from+Kupffer+cells.+Molecular+Therapy.">Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 19, 83-92 (2011).</li><li>Zaiss, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+Heparan+Sulfate+Is+Required+for+Adeno-Associated+Virus+2+but+Dispensable+for+Adenovirus+5+Liver+Transduction+In+Vivo.">Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo.</a> Journal of Virology. 90, 412-420 (2015).</li><li>Delgado, C., Francis, G. E., Fisher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uses+and+properties+of+PEG-linked+proteins.">The uses and properties of PEG-linked proteins.</a> Critical Reviews in Therapeutic Drug Carrier Systems. 9, 249-304 (1992).</li><li>Parveen, S., Sahoo, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicine:+clinical+applications+of+polyethylene+glycol+conjugated+proteins+and+drugs.">Nanomedicine: clinical applications of polyethylene glycol conjugated proteins and drugs.</a> Clin. Pharmacokinet. 45, 965-988 (2006).</li><li>O'Riordan, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylation+of+adenovirus+with+retention+of+infectivity+and+protection+from+neutralizing+antibody+in+vitro+and+in+vivo.">PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo.</a> Human Gene Therapy. 10, 1349-1358 (1999).</li><li>Subr, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coating+of+adenovirus+type+5+with+polymers+containing+quaternary+amines+prevents+binding+to+blood+components.">Coating of adenovirus type 5 with polymers containing quaternary amines prevents binding to blood components.</a> Journal of Controlled Release. 135, 152-158 (2009).</li><li>Kreppel, F., Gackowski, J., Schmidt, E., Kochanek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+genetic+and+chemical+capsid+modifications+enable+flexible+and+efficient+de-+and+retargeting+of+adenovirus+vectors.+Molecular+Therapy.">Combined genetic and chemical capsid modifications enable flexible and efficient de- and retargeting of adenovirus vectors. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 12, 107-117 (2005).</li><li>Corjon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+adenovirus+vectors+to+the+LRP+receptor+family+with+the+high-affinity+ligand+RAP+via+combined+genetic+and+chemical+modification+of+the+pIX+capsomere.">Targeting of adenovirus vectors to the LRP receptor family with the high-affinity ligand RAP via combined genetic and chemical modification of the pIX capsomere.</a> Molecular Therapy: The Journal of the American Society of Gene Therapy. 16 (11), 1813-1824 (2008).</li><li>Prill, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+of+adenovirus+hexon+allow+for+either+hepatocyte+detargeting+or+targeting+with+potential+evasion+from+Kupffer+cells.+Molecular+Therapy.">Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 19, 83-92 (2011).</li><li>Krutzke, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+blood+coagulation+factor+X-binding+to+Ad5+by+position-specific+PEGylation:+Preventing+vector+clearance+and+preserving+infectivity.">Substitution of blood coagulation factor X-binding to Ad5 by position-specific PEGylation: Preventing vector clearance and preserving infectivity.</a> Journal of Controlled Release. 235, 379-392 (2016).</li><li>Prill, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traceless+bioresponsive+shielding+of+adenovirus+hexon+with+HPMA+copolymers+maintains+transduction+capacity+in+vitro+and+in+vivo.">Traceless bioresponsive shielding of adenovirus hexon with HPMA copolymers maintains transduction capacity in vitro and in vivo.</a> PloS One. 9, e82716 (2014).</li><li>Kratzer, R. F., Kreppel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+Purification,+and+Titration+of+First-Generation+Adenovirus+Vectors.">Production, Purification, and Titration of First-Generation Adenovirus Vectors.</a> Methods in Molecular Biology. , 377-388 (2017).</li><li>Blum, H., Beier, H., Gross, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+silver+staining+of+plant+proteins,+RNA+and+DNA+in+polyacrylamide+gels.">Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels.</a> ELECTROPHORESIS. 8, 93-99 (1987).</li></ol>

The authors have nothing to disclose.

The efficiency by which the genetically introduced cysteines can be chemically modified is typically 80-99%, and certain variables influence this efficiency. First, it is paramount that the genetically introduced cysteines do not undergo premature oxidation. While being well-protected in the reducing environment of the producer cells, it is mandatory to provide a non-oxidative environment after releasing vector particles from the producer cells and during chemical modification. To this end, reducing reagents can be used ...

Adenoviruses (Ad), members of the family Adenoviridae, are non-enveloped DNA viruses of which more than 70 types so far have been identified (http://hadvwg.gmu.edu). Depending on hemaglutination properties, genome structure, and sequencing results, the 70 Ad types can be divided into seven species (human adenoviruses A to G)1,2. The human Ad genome is 38 kb in size and encapsulated by an icosahedral nucleocapsid3. Due to their abundance, the capsid protein hexon, penton base, and fiber are all referred to as major capsid proteins. The most abundant and largest capsid protein hexon forms 20 capsid facets, each consisting of 12 hexon homotrimers4,5. Penton, located on each icosahedral edge (vertex), consists of pentamers of a penton base and represents the base for the vertex spike that is built of glycosylated fiber trimers5,6. The native Ad cell entry is basically composed of two major steps. First, the fiber knob binds to the primary receptor. In Ad types from species A, C, E, and F, this is the coxsackie and adenovirus receptor (CAR). This interaction brings the virion into spatial proximity of the cell surface, thereby facilitating interactions between cellular integrins and RGD-motif in the penton base and consequently inducing cellular responses. Second, changes in the cytoskeleton lead to internalization of the virion and transport to the endosome7. Upon partial disassembly in the endosome, the virion is released to the cytoplasm und ultimately travels to the nucleus for replication.
While Ad can be delivered locally (e.g., for genetic vaccination), systemic delivery through the bloodstream as required for onco-virotherapy faces several barriers. While circulating in the bloodstream, injected virions encounter the defense system of the host&#39;s immune system, leading to fast neutralization of the virus-based vectors and rendering Ad-based vectors extremely inefficient in systemic applications. Furthermore, the natural hepatotropism of Ad interferes with systemic delivery and must be resolved to redirect Ad to its new target cells.
Germline-encoded natural IgM antibodies of the innate immune system rapidly recognize and bind highly repetitive structures on the surface of the virion8,9. These immune complexes then activate the classical and non-classical pathways of the complement system, leading to fast complement-mediated neutralization of a large portion of the virions8. A second pathway resulting in major removal of Ad virions is mediated by macrophages10&#160;and associated with acute toxic and haemodynamic side effects11,12. In the case of Ad in particular, Kupffer cells residing in the liver bind to and phagocytically take up the Ad virions via specific scavenger receptors, thereby eliminating them from the blood13,14,15. Specific scavenger receptors have also been identified on liver sinusoidal endothelial cells (LSE cells)16,and LSE cells also seem to contribute to vector elimination17, but to what extent still needs clarification. Furthermore, some Ad types and their&#160;derived vectors are efficiently sequestered by human erythrocytes18&#160;to which they bind via CAR or the complement receptor CR119.&#160;Of note, this sequestering mechanism cannot be studied in the mouse model system as in contrast to human erythrocytes, mouse erythrocytes do not express CAR.
Specific anti-Ad antibodies generated by the adaptive immune system after exposure to Ad either due to previous infections with Ad or after the first delivery in systemic applications raise a further barrier to effective use of Ad vectors, and they should be evaded in efficient systemic delivery.
Finally, the strong hepatotropism of some Ad types (including Ad5) severely hinders application of Ad in systemic therapy. This tropism resulting in hepatocyte transduction is due to the high affinity of the Ad virion to blood coagulation factor X (FX), mediated by the interaction of FX with the Ad hexon protein20,21,22. FX bridges the virion to heparin sulfate glycans (HSPGs) on the surface of hepatocytes20,23,24,25. A crucial factor for this interaction seems to be the specific extent of N- and O-sulfation of the HSPGs in liver cells24, which is distinct from HSPGs on other cell types. In addition to this FX-mediated pathway, recent studies suggest further pathways not yet identified that result in Ad transduction of hepatocytes26,27,28.
Recently, it has been shown that FX is not only involved in hepatocyte transduction of Ad, but also by binding, the virion shields the virus particle against neutralization by the complement system26. Reduction of hepatocyte transduction by preventing FX binding, therefore, would create the unwanted side effect of increasing Ad neutralization via the innate immune system.
A profound knowledge of the complex interactions between vectors and host organisms is therefore necessary to develop more efficient vectors for systemic applications that circumvent the obstacles imposed by the host&#39;s organism.
One strategy that has been originally used for therapeutic proteins has been adapted for Ad vectors to at least partially overcome the above described barriers. Antigenicity and immunogenicity of therapeutic protein compounds could be reduced by coupling to polyethylene glycol (PEG)29,30. Hence, the covalent coupling of polymers such as PEG or poly[N-(2-hydroxypropyl)methacrylamid] (pHPMA) to the capsid surface shields the vector from unwanted vector-host interactions. Commonly, polymer coupling targets &#949;-amine groups from lysine side residues that are randomly distributed on the capsid surface. Vector particles in solution are, due to the hydrophilic nature of the attached polymers, surrounded by a stable water shell that reduces the risk of immune cell recognition or enzymatic degradation. Moreover, PEGylated Ad vectors were shown to evade neutralization by anti-hexon antibodies in vitro and in pre-immunized mice in vivo31. In contrast to genetic capsid modifications, chemical coupling of polymers is performed after production and purification, allowing not only for the use of conventional producer cells and production of high titer vector stocks, but also for simultaneous modification of thousands of amino acids on the capsid surface. However, amine-directed shielding occurs randomly throughout the whole capsid surface, resulting in high heterogeneities and not allowing for modification of specific capsomers. Furthermore, the large polymer moieties required for beneficial effects impair virus bioactivity32.
To overcome these limitations, Kreppel et al.33 introduced a geneti-chemical concept for vector re- and de-targeting. Cysteines were genetically introduced into the virus capsid at solvent-exposed positions like fiber HI-loop33, protein IX34, and hexon35,36. Although not naturally-occurring, cysteine-bearing Ad vectors can be produced at high titers in normal producer cells. Importantly, insertion of cysteines in certain capsomers and in different positions within a single capsomer allows for highly specific modifications of thiol group-reactive moieties. This geneti-chemical approach has been shown to overcome numerous obstacles in Ad vector design. The combination of amine-based PEGylation for detargeting and thiol-based coupling of transferrin to the fiber knob HI-loop has been proven to successfully retarget modified Ad vectors to CAR-deficient cells33. Since hexon is involved in most undesired interactions (neutralizing antibodies, blood coagulation factor FX), thiol-based modification strategies were also applied to hexon. Coupling small PEG moieties to HVR5 of hexon prevented Ad vector particles to transduce SKOV-3 cells in the presence of FX, whereas large PEG moieties increased hepatocyte transduction14,35. Ad vector particles carrying mutations in the fiber knob inhibiting CAR binding and in HVR7 inhibiting binding of FX (and bearing inserted cysteines in HVR1 for position-specific PEGylation) were shown to evade antibody- and complement-mediated neutralization, as well as scavenger receptor-mediated uptake without loss of infectivity. Interestingly, despite a lack of the natural FX shield, PEGylation again improved transduction of hepatocytes as a function of PEG size36. However, it was shown that covalent shielding does have an impact on intracellular trafficking processes. Prill et al. compared irreversible versus bioresponsive shields based on pHPMA and demonstrated that neither the mode of shielding nor co-polymer charge had an impact on cell entry but did affect particle trafficking to the nucleus. Employing a bioresponsive shield with positively charged pHPMA co-polymers allowed for particle trafficking to the nucleus, maintaining the high transduction efficiencies of Ad vectors in vitro and in vivo37.
In summary, these data indicate that, even under the assumption that all vector-host-interactions were known and considered, excessive capsid surface modifications are necessary to overcome the hurdles associated with systemic vector delivery.
Here we provide a protocol to perform site-specific chemical modifications of adenovirus vector capsids for shielding and/or retargeting of adenovirus vector particles and adenovirus-based oncolytic viruses. The concept of this technology is outlined in Figure 1. It allows the shielding of certain capsid regions from unwanted interactions by covalent attachment of synthetic polymers. At the same, it also provides a means to attach ligands and combine shielding and targeting. Using simple chemistry, experimenters will be able to covalently modify adenovirus vector surface with a wide variety of molecules including peptides/proteins, carbohydrates, lipids, and other small molecules. Furthermore, the protocol provides a general concept for the chemical modification of biologically active virus-derived vectors under maintenance of their biological integrity and activity.

<table><tbody><tr><td>&lt;strong&gt;Vector purification and chemical modification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Argon gas</td><td>Air liquide</td><td>local gas dealer</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>Air liquide</td><td>local gas dealer</td><td></td></tr><tr><td>500 mL centrifuge tubes</td><td>Corning</td><td>431123</td><td></td></tr><tr><td>Stericup Express Plus 0.22 &amp;micro;m</td><td>Millipore</td><td>SCGPU02RE</td><td></td></tr><tr><td>Tris(2-carboxyethyl) phosphine (TCEP)</td><td>Sigma-Aldrich</td><td>C4706-10g</td><td></td></tr><tr><td>2 mL (3mL) Norm Ject (syringes)</td><td>Henke Sass Wolf</td><td>4020.000V0</td><td></td></tr><tr><td>Fine-Ject needles for single use (yellow 0.9 x 40 mm)</td><td>Henke Sass Wolf</td><td>4710009040</td><td></td></tr><tr><td>Caesium chloride 99.999% Ultra Quality</td><td>Roth</td><td>8627.1</td><td></td></tr><tr><td>Silica gel beads</td><td>Applichem</td><td>A4569.2500</td><td></td></tr><tr><td>Methoxypolyethylene glycol maleimide - 750 (PEG mal-750)</td><td>Iris Biotech</td><td></td><td>store in silica gel beads at -80 &amp;deg;C</td></tr><tr><td>13.2 mL Ultra Clear Ultracentrifuge Tubes</td><td>Beckman Coulter</td><td>344059</td><td>only open in hood</td></tr><tr><td>PD-10 size exclusion chromatography column</td><td>GE Healthcare</td><td>17-0851-01</td><td>store at 4 &amp;deg;C</td></tr><tr><td>Hepes</td><td>AppliChem</td><td>A1069.1000</td><td></td></tr><tr><td>SDS Ultrapure</td><td>AppliChem</td><td>A1112,0500</td><td></td></tr><tr><td>Glycerol</td><td>AppliChem</td><td>A1123.1000</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material for cell-culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DPBS</td><td>PAN Biotech</td><td>P04-36500</td><td></td></tr><tr><td>DMEM</td><td>PAN Biotech</td><td>P04-03590</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>PAN Biotech</td><td>P10-0231SP</td><td></td></tr><tr><td>FBS Good</td><td>PAN Biotech</td><td>P40-37500</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>PAN Biotech</td><td>P06-07100</td><td></td></tr><tr><td>Biosphere Filter Tips (various sizes)</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>Serological Pipettes (various sizes)</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>reaction tubes (various sizes)</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>TC plates 15cm</td><td>Sarstedt</td><td>83.3903</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material for silver staining protocol&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Methanol</td><td>J.T.Baker</td><td>8045</td><td></td></tr><tr><td>Ethanol absolute</td><td>AppliChem</td><td>1613,2500PE</td><td></td></tr><tr><td>Acetic Acid</td><td>AppliChem</td><td>A0820,2500PE</td><td></td></tr><tr><td>Formaldehyde 37%</td><td>AppliChem</td><td>A0877,0250</td><td></td></tr><tr><td>Ethanol absolute</td><td>AppliChem</td><td>A1613,2500PE</td><td></td></tr><tr><td>Sodium thiosulfate</td><td>AppliChem</td><td>1,418,791,210</td><td></td></tr><tr><td>Silver nitrate</td><td>AppliChem</td><td>A3944.0025</td><td></td></tr><tr><td>Sodium carbonate</td><td>AppliChem</td><td>A3900,0500</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Special Lab Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Desiccator</td><td>Nalgene</td><td>5311-0250</td><td></td></tr><tr><td>Megafuge 40</td><td>Heraeus</td><td></td><td></td></tr><tr><td>Roter for Megafuge TX750 + Adapter andLlids for 500 mL tubes</td><td>Heraeus</td><td></td><td></td></tr><tr><td>Water bath</td><td>Conventional</td><td></td><td></td></tr><tr><td>Ultracentrifuge &lt;em&gt;e.g. &lt;/em&gt;Optima XPN-80</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>suitable Ultrazentrifuge Rotor&lt;em&gt; e.g. &lt;/em&gt;SW41</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>pH -Meter</td><td>Conventional</td><td></td><td></td></tr><tr><td>Stand with clamps</td><td>Conventional</td><td></td><td></td></tr><tr><td>Goose neck lamp</td><td>Conventional</td><td></td><td></td></tr><tr><td>Over-head rotor</td><td>Conventional</td><td></td><td></td></tr><tr><td>Thermal Block</td><td>Conventional</td><td></td><td></td></tr><tr><td>Photometer (OD 260)</td><td>Conventional</td><td></td><td></td></tr></tbody></table>

combined genetic and chemical capsid modifications of adenovirus-based gene transfer vectors for shielding and targeting

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

Figure 2 shows examples of the cytopathic effect (CPE) on 293 (HEK 293) cells that indicates successful vector production. Cells should show morphology (Figure 2C) 40-48 hours after inoculation with the virus vector. The right timepoint for harvesting is crucial for not losing virus particles by cell lysis and preventing oxidation of the genetically introduced thiol groups. If vector particles are released into the medium by cell...

Watch this Scientific Journal Video about Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting at JoVE.com

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host ...

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Molecular Biology

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Unit 1

DNA, Cells, and Evolution

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8.3K Views.  University Witten/Herdecke. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van gentherapie, oncolyse en genetische vaccinatie. Het vergemakkelijkt het begrijpen en moduleren van vectorgasthermotoren interacties van adenovirus genoverdrachtvectoren, het meest gebruikte vectortype in klinische studies up-to-date. Het belangrijkste voordeel van deze techniek is dat adenovirus capsulates specifiek kunnen worden gewijzigd op een zeer gedefinieerde manier op geselecteerde posities en in vari...

Video: Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Cancer Research

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Immunology and Infection

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Bioengineering

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Neuroscience

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by encouraging clinical data and the approval of several AAV gene therapies. Two major reasons for the success of AAV vectors are (i) the prior isolation of various naturally occurring viral serotypes with distinct properties, and (ii) the subsequent establishment of powerful technologies for their molecular engineering and repurposing in high throughput. Further boosting the potential of these techniques are recently implemented strategies for barcoding selected AAV capsids on the DNA and RNA level, permitting their comprehensive and parallel in vivo stratification in all major organs and cell types in a single animal. Here, we present a basic pipeline encompassing this set of complementary avenues, using AAV peptide display to represent the diverse arsenal of available capsid engineering technologies. Accordingly, we first describe the pivotal steps for the generation of an AAV peptide display library for the in vivo selection of candidates with desired properties, followed by a demonstration of how to barcode the most interesting capsid variants for secondary in vivo screening. Next, we exemplify the methodology for the creation of libraries for next-generation sequencing (NGS), including barcode amplification and adaptor ligation, before concluding with an overview of the most critical steps during NGS data analysis. As the protocols reported here are versatile and adaptable, researchers can easily harness them to enrich the optimal AAV capsid variants in their favorite disease model and for gene therapy applications.

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants

AAV peptide display library generation and subsequent validation through the barcoding of candidates with novel properties for the creation of next-generation AAVs.

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by ...

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an ...

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

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